Project Summary/Abstract The largest risk factor for developing chronic disease, including neurodegenerative diseases, such as Alzheimer?s disease (AD), is age. There is now abundant evidence that aging processes can be driven by DNA damage and a major pathologic consequence of DNA damage and its erroneous repair is DNA mutation, from base substitutions to very large chromosomal alterations. Elevated chromosomal aneuploidy has been reported in human neuronal cells in the brain of AD, these results are controversial and have been suggested to possibly be false positives of the assay used, i.e., Fluorescent in situ Hybridization (FISH). Somatic genomic mosaicism analysis thus far has been mostly directed to neurons with little focus on glial cells, which are emerging as crucial players mediating development and homeostasis of the central nervous system. In AD growing interest in glia is primarily fueled by genome-wide association study (GWAS) discovery of risk loci in genes related to the innate immune system and by the recognized importance of reactive glia in clinical appearance and progression of cognitive decline in AD. Previous work in Project 2 of the PPG has uncovered an age-related increase of chromosome aneuploidies in glial cells but not neurons of the cerebral cortex of mice. This led to our current hypothesis that non-neuronal (NeuN-neg) brain cells are particularly susceptible to age-related large-scale genomic instability resulting in loss of their physiological functions. Ultimately NeuN-neg glial cells that acquired large scale DNA damage could contribute to AD and age-related dementias. To test this hypothesis, we propose two specific aims. Aim 1 will provide the landscape of chromosome instability in glial cells in the cortex and hippocampus of sporadic Alzheimer?s disease brains. Using single-cell low-coverage whole genome sequencing (scL-WGS) we will determine whether aneuploidy or other forms of large-scale copy number alterations (CNAs) accumulate during human aging in brain regions associated with AD and AD-type dementias in NeuN-neg as compared to NeuN+ cellular genomes. All samples will also be analyzed by Interphase Fluorescent in situ Hybridization (iFISH), a sensitive custom 4-color interphase FISH assay that provides enhanced sensitivity and specificity for the measurement of ploidy changes and aneuploidy. In Aim 2 we will establish the transcriptional heterogeneity of glial cells in AD patients and controls and their relationship to large-scale genomic instability in the brain. Building on our recently developed multicolor interphase DNA-RNA-FISH assay (iDR-FISH) we propose to use spatially-resolved transcriptomics to study genomic instability in the context of the brain microenvironment in the AD and control transentorhinal cortex (TEC). Globally these studies will provide unprecedented knowledge of large-scale genomic instability in the aged and diseased brain and its functional link to neurodegeneration.